MicroRNAs (miRNAs) commonly repress gene expression by binding to the 3'untranslated region of target mRNAs, and thousands of mRNAs have been predicted to be targets of hundreds of miRNAs in animals. Despite intense studies in the past decade, the majority of regulatory miRNA::mRNA interactions for a broad spectrum of physiological functions remain to be elucidated. In C. elegans, genetic analyses have only linked a small percentage of miRNAs or miRNA families to prominent roles in development. The difficulty encountered in functional studies of miRNAs may be attributed in part to that multiple miRNAs from different families may collaborate to regulate a set of target genes for specific physiological functions. We may also hypothesize that a large number of miRNAs may function in cellular processes involved in animals'responses to environmental changes such as nutrient availability, stress conditions and pathogen infection. Our goal is to gain a global view of dynamic miRNA::target interactions in different tissues and under different physiological conditions to obtain insights regarding miRNA-mediated functions in stress and infection responses. We propose to combine novel systematic approaches with individual gene- based analysis to tackle the problem, using the nematode C. elegans as a model system. We will first evaluate physiological functions of miRNAs by disrupting activities of all miRNAs in selected tissues and examine the developmental and non- developmental consequences, including responses to stresses and infections. We will then identify and analyze miRNA::target interactions in selected tissues during development by applying AIN-2 immunoprecipitation, high-throughput RNA analysis, and computational methods. This approach will further be used to identify dynamic miRNA::target interactions during animals'responses to food deprivation, pathogen infection and other stresses. Finally, we will carry out experiments to verify miRNA::target interactions for selected pairs and analyze their functions in stress and pathogen responses. The results of this study should provide important insights regarding important functions of miRNA regulations and mechanisms of animals'defense against environmental stresses and infections. PUBLIC HEALTH RELEVANCE: microRNAs are evolutionarily conserved small, non-coding RNAs that have been shown to play critical roles in a broad aspect of cellular and developmental processes, including many human diseases such as cancers, cardiac and neurological diseases, as well as animal's responses to various stresses. Research under this grant is expected to make a solid contribution by providing new insights into miRNA functions in stress responses and development.